Novel combination of benzoquinazoline antifolates and protecting agents

ABSTRACT

The use of a protecting agent, for example a folate derivative such as folic acid or leucovorin, in combination with a non-competitive folic acid analogue, for example benzoquinazoline derivatives, for use in reducing the side effects associated with the administration of such non-competitive folic acid analogues, and pharmaceutical formulations comprising such combinations are disclosed.

[0001] The present invention relates to novel combinations ofnon-competitive folic acid analogues with protecting agents, and tomethods of treatment using these combinations.

[0002] European Patent Application 0505640 provides a method forimproving the therapeutic utility of GAR-transformylase inhibitors andother antifolates by co-administering a folate binding protein bindingagent to the host undergoing treatment.

[0003] Thymidylate synthase is an enzyme catalysing the terminal step inthe de novo synthesis of thymidylate required for DNA synthesis. It hasbeen postulated that inhibitors of this enzyme may be expected to haveanti-tumour activity and it has been reported (Jones et al, J. Med.Chem. 1986, 29, 468) that the in-vivo antitumour activity ofN¹⁰-propargyl-5,8-dideazafolic acid arises solely from its inhibitoryeffect on this enzyme.

[0004] PCT publication WO/91/19700 discloses the compound of the formula(I)

[0005] or a salt thereof, wherein the dotted line represents a single ordouble bond.

[0006] R¹ is C₁₋₄ alkyl or amino optionally substituted by a C₁₋₄ alkylC₁₋₅ alkanoyl or benzyl group;

[0007] R², R³, R⁴ and R⁵ are the same or different and each is selectedfrom hydrogen, phenyl, halo, nitro.

[0008] a group S(O)_(n) R⁸ wherein n is the integer 0, 1 or 2 and R⁸ ishalo or is C₁₋₄ alkyl or a group NR⁹R¹⁰ wherein R⁹ and R¹⁰ are bothhydrogen,

[0009] a group NR¹¹R¹² wherein R¹¹ and R¹² are the same or different andeach is hydrogen or C₁₋₄ alkyl.

[0010] a group OR¹³ wherein R¹³ is hydrogen or C₁₋₄ alkyl optionallysubstituted by halo;

[0011] a C₁₋₄ aliphatic group optionally substituted by a group OR¹⁴ orNR¹⁴R¹⁵ wherein R¹⁴ and R¹⁵ are the same or different and each ishydrogen or C₁₋₄ alkyl:

[0012] or two of R² to R⁵ are linked together to form a benzo group.

[0013] or one of R² to R⁵ is a group —X—Y—R¹⁶ wherein X is CH₂, NR¹⁷, COor S(O)_(m) and m is 0, 1 or 2 and R¹⁷ is hydrogen or a C₁₋₄ aliphaticgroup and Y is CH₂, O, NR¹⁷′ or S(O)_(m′) wherein m′ is 0, 1 or 2 andR¹⁷ is hydrogen or a C₁₋₄ aliphatic group provided that X and Y are onlythe same when each is CH₂, or —X—Y— is a group —O—. —NR¹⁷—, —CH═CH— or—N═N— wherein R¹⁷ is as hereinbefore defined, R¹⁶ is a C₁₋₄ aliphaticgroup or a 5- or 6-membered aromatic ring optionally substituted by agroup R¹⁸ at a position at least one carbon atom removed from thatlinked to Y, the 5- or 6-membered ring being optionally furthersubstituted by a halo atom; and R¹⁸ is halo, C₁₋₄ alkoxy, nitro,nitrile, C₁₋₄ alkyl optionally substituted by halo, halo or a groupCOR¹⁹ wherein R¹⁹ is hydroxy, C₁₋₄ alkoxy or C₁₋₆ alkyl optionallysubstituted by one or two carboxyl groups or C₁₋₁₂ esters thereof or R¹⁹is a group NR²⁰R²¹ wherein R²⁰ and R²¹ are the same or different andeach is hydrogen or C₁₋₄ alkyl optionally substituted by hydroxy or R¹⁹is an amino acid group or an ester thereof in which the first nitrogenatom of the amino acid group may be linked to the 5- or 6-memberedaromatic ring to form a further 5- or 6-membered heterocyclic ring orR¹⁹ is an C₂₋₃ alkylene croup linked to the 5- or 6-membered aromaticring to form a further 5- or 6-membered ring;

[0014] R⁶ and R⁷ are the same or different and each is hydrogen, C₁₋₄alkyl optionally substituted by hydroxy or C₁₋₄ alkoxy or together forma benzo group;

[0015] provided that at least one of R² to R⁷ is other than hydrogen andthat R⁴ is not methoxy when R¹ is hydroxy or methyl is an inhibitor ofthe enzyme thymidylate synthase and has anti-tumour activity.

[0016] By the term halo is meant fluoro, bromo, chloro and iodo.

[0017] By the term C₁₋₄ aliphatic group is meant a C₁₋₄ alkyl, alkenyl,or alkynyl group.

[0018] By the term amino acid group is meant naturally occurring aminoacids.

[0019] Preferred amino acid groups include glycine, glutamic acid andpolyglutamic acid groups.

[0020] When the amino acid group is linked to the 5- or 6-memberedaromatic ring, this is via a carbon atom of the aromatic ring adjacentto carbon to which COR¹⁹ is attached.

[0021] Preferably the dotted line is a double bond.

[0022] Suitable substituents for the aromatic ring R¹⁶ include halo,C₁₋₄ alkyl and C₁₋₄ alkoxy each optionally substituted by one to fivehalo atoms. Most suitably there are one or two substituents selectedfrom fluoro, chloro, methyl, trifluoromethyl and methoxy, and preferablyfluoro, or no substituents on the aromatic ring. In one preferredembodiment, —X—Y—R¹⁶ is a group

[0023] wherein R¹⁸ is as hereinbefore defined and preferably a groupCOR¹⁹ as hereinbefore defined and R²² is hydrogen or fluoro.

[0024] In a further preferred embodiment X—Y—R¹⁶ is a group

[0025] wherein H₂NR^(19a) is a glutamic or polyglutamic acid group and Zis CH₂, S or O.

[0026] Suitably, R¹ is an amino group optionally substituted by one ortwo methyl or ethyl groups or R¹ is a methyl or ethyl group. PreferablyR¹ is an amino or methyl group.

[0027] Suitably, at most only three, and preferably at most only two, ofR² to R⁵ are other than hydrogen and each is independently selected fromhydrogen, halo, hydroxy, nitro, C₁₋₃ alkyl optionally substituted byhydroxy or C₁₋₂ alkoxy, C₁₋₃ alkoxy, amino optionally substituted by oneor two methyl or ethyl groups, or a group S(O)_(n) R²³ wherein n is 0, 1or 2and R²³ is a C₁₋₄ alkyl group or an amino group optionallysubstituted by one or two methyl or ethyl groups, or one of R² to R⁵ isa group —X—Y—R²⁴ where R²⁴is a group

[0028] wherein R¹⁸, R^(19a), R²² and Z are as hereinbefore defined. Inone preferred embodiment R¹⁸ is nitro or a group

[0029] wherein R²⁵, R²⁶ and R²⁷ are the same or different and each ishydrogen or a C₁₋₄ alkyl group and t is an integer from 0 to 6.Preferably R²⁵, R²⁶ and R²⁷ are hydrogen and t is 0. Preferably Z is CH₂or S.

[0030] Preferably one of R² to R⁵ is a group —X—Y—R²⁴ as hereinbeforedefined. Preferably R³ is a group —X—Y—R²⁴.

[0031] Suitably R⁶ and R⁷ are the same or different and each ishydrogen, methyl, ethyl or methyl substituted by bromo, hydroxy ormethoxy. Preferably R⁷ is hydrogen and R⁶ is methyl.

[0032] Preferably —X—Y— is a group —SO₂NR¹⁷— or CH₂NR¹⁷ wherein R¹⁷ isas hereinbefore defined.

[0033] Suitably R¹⁷ is hydrogen or a C₁₋₄ alkyl or alkenyl group andpreferably R¹⁷ is hydrogen or methyl.

[0034] A further group of compounds of the formula I is that of theformula (II)

[0035] or a salt thereof, wherein R¹, R⁶, R⁷ and the dotted line are ashereinbefore defined and R²⁸ to R³¹ are the same or different and eachis selected from hydrogen, halo, nitro, a group S(O)_(n)R⁸, a groupNR¹¹R¹², a group OR¹³, or a C₁₋₄ aliphatic group optionally substitutedby a group OR¹⁴ or NR¹⁴R¹⁵ wherein R⁸, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵hereinbefore defined, provided that R²⁸ to R³¹ are not all hydrogen andthat R³⁰ is not methoxy wherein R¹ is hydroxy or methyl.

[0036] A preferred group of compounds of the formula (I) is that of theformula (III):

[0037] or a salt thereof, wherein R¹, R⁶ and R⁷ are as hereinbeforedefined and R³² to R³⁵ are the same or different and one is a groupX—Y—R¹⁶ and the others are the same or different and each is selectedfrom hydrogen, halo, nitro, a group S(O)_(n)R⁸, a group NR¹¹R¹² a groupOR¹³ or a C₁₋₄ aliphatic group optionally substituted by a group OR¹⁴ orNR¹⁴R¹⁵, wherein X, Y, R⁸, R¹¹, R¹², R¹³,R¹⁴, R¹⁵ and R¹⁶ are ashereinbefore defined.

[0038] A further preferred group of compounds of the formula (I) is thatof the formula (IV):

[0039] wherein R¹, R⁶, R⁷ and R³² to R³² are as hereinbefore defined.

[0040] Preferably R³³ is a group X—Y—R¹⁶ as hereinbefore defined.

[0041] Preferred compounds of the formula (I) include:

[0042] 3-Amino-9-bromobenzo[f]quinazolin-1(2H)-one

[0043] 3-Amino-9-ethynylbenzo[f]quinazolin-1(2H)-one

[0044]N-(4-((3-Amino-1,2,5,6-tetrahydro-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamicacid

[0045]N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L-glutamicacid

[0046]N-(4-((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamicacid

[0047]N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamicacid

[0048]N-(4(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)benzoyl)-L-glumaticacid

[0049](S)-2-(5-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino-1-oxo-2-isoindolinyl)glutaricacid

[0050] 9-((4-Acetylanilino)methyl)-3-methylbenzo[f]quinazolin-1(2H)-one

[0051] 3-Methyl-9-((4-nitroanilino)methyl)benzo[f]quinazolin-1(2H)-one

[0052]N-(4-(((3-Amino-1,2-dihydro-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)benzoyl)-L-glutamicacid

[0053] 3-Amino-9-((4-nitroanilino)methyl)benzo[f]quinazolin-1(2H)-one

[0054] 9-((4-Acetylanilino)methyl)-3-aminobenzo[f]quinazolin-1(2H)-one

[0055](RS)-2-(2-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-2-oxoethyl)glutaricacid

[0056]Ethyl4-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-4-oxobutyrate

[0057]4-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)phenyl)-4-oxobutyricacid

[0058]N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)glycine

[0059] EthylN-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)glycinate

[0060] Certain compounds of the formula (I) contain asymmetric carbonatoms and are, therefore, capable of existing as optical isomers. Theindividual isomers and mixtures of these are included within the scopeof the present invention.

[0061] It has now been found that the compounds of formula (I) can beused in combination with a protecting agent described hereinafter toprevent or decrease unwanted side effects of these compounds.

[0062] The present invention relates to the administration of thecompounds of formula (I) in combination with a protecting agent whichblocks the intestinal toxicity of the compounds of formula (I) withoutblocking their activity. Suitable protecting agents are folates, forexample, folic acid, tetrahydrofolate, 5-formyltetrahydrofolate(leucovorin) or 5-methyltetrahydrofolate. Other protecting agentsinclude thymine or thymidine. The protecting agent may be administeredas an oral formulation. It is within the scope of the invention toadminister the protecting agent before, during or after theadministration of a compound of formula (I). Preferably, the protectingagent will be given slightly before administration of a compound offormula (I), i.e. between 15 minutes and one hour before the compound offormula (I).

[0063] The present invention also relates to combination therapycomprising the administration of non-competitive folic acid analogues incombination with protecting agents as defined above. This allows the useof the non-competitive folic acid analogues at higher doeses than couldbe used in the absence of the protecting agent as defined above, andtherefore higher, more potent doses can be achieved while minimizinghost toxicity. The presence of the protecting agent reduces the toxicityof the non-competitive folic acid analogues at the doses utilized in theabsence of the protecting agent (i.e. less toxicity at the same dose inthe presence of a protecting agent).

[0064] Non-competitive folic acid analogues are defined as structuralanalogues of folic acid (including but not limited to thebenzoquinazoline compounds of formula I, e.g., compounds of example 1 or2) expressing classical non-competitive inhibition kinetics (vs. thefolate substrate) on the target enzyme (e.g., thymidylate synthase,glycineamide ribonucleotide transformylase or dihydrofolate reductase).Alternatively, the non-competitive inhibitors of the present inventionare defined as structural analogues of folic acid (as defined above)which retain their tumour cell kill in the presence of the folateprotecting agents defined above, on the target enzyme. Thenon-competitive inhibitor may been given as an intravenous orintraperitoneal bolus or infusion and the protecting agent may be givenas an intravenous or intraperitoneal bolus infusion or as an oralformulation. For a definition of classical non-competitive inhibitionsee I. H. Segal (1975) Enzyme kinetics, John Wiley and Sons, which isincorporated herein by reference.

[0065] More specifically, protecting agents selected from folic acid,tetrahydrofolate. 5-formyltetrahydrofolate (leucovorin) or5-methyltetrahydrofolate can block the intestinal toxicity of compoundsof formula (I) without blocking their anti-tumor activity.

[0066] Preferably the non-competitive, folic acid analogue (i.e.,non-competive inhibitor) is a compound of formula (I) and the protectingagent is folic acid or 5-formyltetrahydrofolate. More preferably, thenon-competitive, folic acid analogue (non-competitive inhibitor) is acompounds selected from(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid.N-(4-(((1,2-dihydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamicacid, orN-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo(f)quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamicacid and the protecting agent which is used in combination is folic acidor 5-formyltetrahydrofolate. Most preferably, the compound(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxoxbenxo(f)quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid is utilized in combination with folic acid or5-formyltetrahydrofolate.

[0067] The salts of the compounds of the present invention can also beused in combination with the protecting agent or salts thereof.

[0068] Suitable salts of the compounds of the present invention or ofthe protecting agents may comprise acid addition salts derived from anamino group or anionic species derived from a compound of formula (I) orprotecting agent, for example when this is substituted by a carboxygroup, and a cation. In both types of salts, the therapeutic activityresides in the moiety derived from the compounds defined herein and theidentity of the other component is of less importance although fortherapeutic and prophylactic purposes it is, preferably,pharmaceutically acceptable to the patient. Examples of pharmaceuticallyacceptable acid addition salts include those derived from mineral acids,such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitricand sulphuric acids, and organic acids, such as tartaric, acetic,trifluoroacetic, citric, malic, lactic, fumaric, benzoic, glycollic,gluconic, succinic and methanesulphonic and arylsulphonic, for examplep-toluenesulphonic, acids. Examples of salts comprising an anionicspecies derived from a compound of formula (I) and a cation includeammonium salts, alkali metal salts, such as sodium and potassium salts,alkaline earth salts, such as magnesium and calcium salts and saltsformed with organic bases, for example, amino salts derived from mono-,di- or tri-(lower alkyl) or (lower alkanol)amines, such astriethanolamine and diethylamino-ethylamine, and salts with heterocyclicamines such as piperidine, pyridine, piperazine and morpholine. Thepharmaceutically acceptable salts together with the salts which are notthus acceptable have utility in the isolation and/or the purification ofthe compounds of the invention, and the pharmaceutically unacceptablesalts are also useful in being convertible to the pharmaceuticallyacceptable salts by techniques well known in the art.

[0069] The route by which the compound or salt of the present inventionis administered to the animal may be oral, topical, parenteral(including subcutaneous, intradermal, intramuscular, intravenous orrectal). If the compound or salt is presented in the form of apharmaceutical formulation, which is preferred, then the actualformulation employed will of course depend on the route ofadministration elected by the physician or veterinarian. For example, iforal administration is preferred, then the pharmaceutical formulationemployed is, preferably, one which is suitable for such a route.

[0070] A therapeutically effective amount of a compound or salt of thepresent invention will depend upon a number of factors including, forexample, the age and weight of the animal, the precise conditionrequiring treatment and its severity, the nature of the formulation, andthe route of administration, and will ultimately be at the discretion ofthe attendant physician or veterinarian. However, an effective amount ofa non-competitive folic acid analogue of the present invention (asdescribed hereinbefore) to be used in combination with a protectingagent (as described hereinbefore) for the treatment of neoplasticgrowth, for example colon or breast carcinoma will generally be in therange of 0.1 to 100 mg/kg body weight of recipient (mammal) per day andmore usually in the range of 1 to 50 mg/kg body weight per day. Thus,for a 70 kg adult mammal, the actual amount per day would usually befrom 70 to 3500 mg and this amount may be given in a single dose per dayor more usually in a number (such as two, three, four, five or six) ofsub-doses per day such that the total daily dose is the same. Aneffective amount of a salt of the present invention may be determined asa proportion of the effective amount of the compound per se.

[0071] An effective amount of a compound of the present invention forthe treatment of disorders of the immune system (e.g. thermatoidarthritis and tissue rejection) and related diseases such as psoriasis,will generally be in the range of 0.5-10 mg/kg body weight of recipient(mammal) per day. Thus for a 70 kg adult human, the actual amount perday would usually be from about 5-700 mg per day and this amount may begiven in a single dose per day or more usually dosing would beintermittent e.g. 12 hourly intervals or weekly. An effective amount ofa salt of the present invention may be determined as a proportion of theeffective amount of the compound per se.

[0072] An effective amount of a compound of the present invention forthe treatment of bacterial and fungal infections is in the range of0.1-100 mg/kg bodyweight of recipient (mammal) per day and preferably inthe range of 1-50 m/kg body weight per day. Thus, for a 70 kg adultmammal, the actual amount per day is from 70-3500 mg and this amount maybe given in a single dose per day or more preferably in a number (suchas two, three, four, five or six) of sub-doses per day such that thetotal daily dose is the same. An effective amount of a salt of thepresent invention may be determined as a proportion of the effectiveamount of the compound per se.

[0073] An effective amount of a protecting agent (e.g., folic acid) ofthe present invention for use in combination with a non-competitive,folic acid analogue (including but not limited to the compounds of thepresent invention as described herein) is in the range of 1-300 mg/kgbodyweight of recipient (mammal) per day and preferably in the range of5-50 mg/kg body weight per day. Thus, for a 70 kg adult mammal, theactual amount per day is from 350-3500 mg and this amount may be givenin a single dose per day or more preferably in a number (such as two,three, four, five or six) of sub-doses per day such that the total dailydose is the same.

[0074] The treatment of neoplastic growth with a compound of the presentinvention may at times require the administration to the animal of anantidote or rescue agent, e.g. thymidine.

[0075] The compounds, per se, of the present invention which can be usedin combination with a protecting agent as described herein include, butare not limited to, those disclosed in PCT publication WO/91/19700. Theprotecting agents of the present invention are readily available andwell known to those skilled in the art.

[0076] Whilst it is possible for the compounds or salts of the presentinvention to be administered as the raw chemical, it is preferred topresent them in the form of a pharmaceutical formulation.

[0077] Accordingly, the present invention further provides apharmaceutical formulation, for medicinal application, which comprises acompound of the present invention and a protecting agent orpharmaceutically acceptable salts thereof, as hereinbefore defined, incombination with one or more pharmaceutically acceptable carrierstherefor, and optionally one or other therapeutic agredients.

[0078] The pharmaceutical formulation may optionally contain othertherapeutic agents that may usefully be employed in conjunction with thecompound or salt of the present invention, for example a pyrimidinenucleoside transport inhibitor that is capable of enhancing theantineoplastic activity of the compounds and salts of the presentsinvention. The expression “pharmaceutically acceptable” as used hereinin relation to the carrier is used in the sense of being compatible withthe compound or salt of the invention employed in the formulation andwith any other therapeutic agent that may be present, and not beingdetrimental to the recipient thereof. The carrier itself may constituteone or more excipients conventionally used in the art of pharmacy thatenable the compound or salt of the present invention and any othertherapeutic agent that may be present, to be formulated as apharmaceutical formulation.

[0079] The pharmaceutical formulations of the present invention includethose suitable for oral, parenteral (including subcutaneous,intradermal, intramuscular and intravenous) and rectal administrationalthough the most suitable route will probably depend upon, for example,the condition and identity of the recipient. The formulations mayconveniently be presented in unit dosage form and may be prepared by anyof the methods will known in the art of pharmacy. All methods includethe step of bringing into association the active ingredient, i.e., thecompound or salt and protecting agent of the present invention, with thecarrier.

[0080] In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with aliquid carrier or, a finely divided solid carrier or both, and then, ifnecessary, forming the associated mixture into the desired formulation.The pharmaceutical formulations of the present invention suitable fororal administration may be presented as discrete units, such as acapsule, cachet, tablet, or lozenge, each containing a predeterminedamount of the active ingredient, as a powder or granules; as a solutionor a suspension in an aqueous liquid or a non-aqueous liquid such as asyrup, elixir or a draught, or as an oil-in water liquid emulsion or awater-in-oil liquid emulsion. The formulation may also be a bolus,electuary or paste.

[0081] Generally, a tablet is the most convenient pharmaceuticalformulation suitable for oral administration. A tablet may be made bycompressing or moulding the active ingredient with the pharmaceuticallyacceptable carrier. Compressed tablets may be prepared by compressing ina suitable machine the active ingredient in a free-flowing form, such asa powder or granules, in admixture with, for example, a binding agent,an inert diluent, a lubricating agent, a disintegrating agent and/or asurface active agent. Moulded tablets may be prepared by moulding in asuitable machine a mixture of the powdered active ingredient moistenedwith an inert liquid diluent. The tablets may optionally be coated orscored and may be formulated so as to provide slow or controlled releaseof the active ingredient.

[0082] The pharmaceutical formulations of the present invention suitablefor parenteral administration include aqueous and non-aqeuous sterileinjection solutions which may contain, for example, an anti-oxidant, abuffer, a bacteriostat and a solution which renders the compositionisotonic with the blood of the recipient, and aqueous and non-aqueoussterile suspensions which may contain, for example, a suspending agentand a thickening agent. The formulations may be presented in unit-doseor multi-dose containers, for example sealed ampoules and vials, and maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water for injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

[0083] The pharmaceutical formulations of the present invention suitablefor rectal administration may be presented as a suppository containing,for example, cocoa butter and polyethylene glycol.

[0084] Compounds and salts of formula (I) have anti-neoplastic activityin the human colon SW480 adenocarcinoma cell culture cytotoxicity testsin which representative compounds of the present invention have beenshown to be active, and in human breast MCF7 adenocarcinoma cellculture. It has thus been established that compounds of the presentinvention are able to inhibit neoplastic growth. Therefore, compoundsand salts of the present invention are of use in medicine and inparticular in the treatment of neoplastic growth, including solidtumours such as melanoma breast and colon tumours in mammals.Accordingly, the present invention yet further provides a method for thetreatment of susceptible malignant tumours and leukemia in a animal.e.g., a mammal, which comprises administering to the animal atherapeutically effective amount of a compound or salt of the presentinvention in combination with a protecting agent. In the alternative,there is also provided a compound or salt of the present invention incombination with protecting agent. In the alternative, there is alsoprovided a compound or salt of the present invention in combination witha protecting agent for use in medicine and in particular for use in thetreatment of a neoplastic growth, e.g., malignant tumours.

[0085] The present invention also provides the use of a compound offormula (I) or a salt therof in combination with a protecting agent forthe manufacture of a medicament for the treatment of neoplastic growth.

[0086] The animal requiring treatment with a compound or salt of thepresent invention in combination with a protecting agent is usually amammal, such as a human being.

[0087] The following examples illustrate the preparation andpharmacological properties of representative compounds which are usefulin the present invention and which demonstrate the invention. Theseexamples are illustrations only and should not be construed as limitingor narrowing the scope of the invention.

EXAMPLE 1(S)-2-(5-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)-amino)-1-oxo-2-isoindolinyl)glutaricacid (Compound A)

[0088] A. Diethyl (S)-2-(4-nitrophthalimido)glutarate

[0089] Diisopropylethylamine (24 ml, 0.138 mole) (Aldrich) was added toa suspension of 4-nitrophthalic anhydride (25 g, 0.13 mole) (TokyoKasei) and L-glutamic acid diethyl ester hydrochloride (35 g, 0.146mole) (Aldrich) in toluene (130 ml). The reaction mixture was stirred atreflux utilizing a Dean-Stark trap for 2.5 hours. After cooling, thesolution was diluted with diethyl ether (300 ml), washed with water (75ml), saturated NaHCO₃ solution (50 ml), dried (MgSO₄), and concentratedin vacuo at 70° C. to give diethyl (S)-2-(4-nitrophthalimido) glutarateas an oil that solidified to a white solid on standing (35.8 g).M.P.=65.5-66.5° C.

[0090] B. Diethyl (S)-2-(4-aminophthalimido)glutarate

[0091] A suspension of diethyl (S)-2-(4-nitrophthalimido)glutarate (35.6g, 94.1 mmol) and 10% palladium on carbon (0.5 g) (Aldrich) in ethanol(200 ml) was shaken under a hydrogen atmosphere (40-50 psi) for 26hours. The solution was filtered through celite and concentrated invacuo. The residue was purified by chromatography on silica gel (250 g)eluting with diethyl ether:hexane (4:1) to give diethyl(S)-2-(4-aminophthalimido)glutarate as a viscous yellow oil (29.1 g).

[0092] C. Diethyl (S)-2-(5-amino-1-oxo-2-isoindolinyl)glutarate

[0093] A solution of diethyl (S)-2-(4-aminophthalimido)glutarate (10.5 g30.2 mmol) in ethanol (150 ml) was cooled in an acetonitrile/CO₂ bath.Concentrated HCl (25 ml) was added followed by 30 mesh granular Zn (10.5g, 0.161 mole) (Fisher) when the internal temperature has reached −40°C. The reaction mixure was stirred 1.5 hours at this temperature and afurther 1 hour at −10° C. The excess of Zn was filtered from thesolution, 10% palladium on carbon (1.0 g) was added, and the solutionshaken under hydrogen at (30-50 psi) overnight. The catalyst was removedby filtration through celite and the filtrate concentrated in vacuo. Theresidue was absorbed onto silica gel (15 g) and purified bychromatography on silica gel (440 g) eluting with ethylacetate:methylene chloride (1:14 g).

[0094] D 9-Bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one

[0095] To a hot solution of 3,9-dimethylbenzo[f]quinazolin-1(2H)-one(4.00 g, 17.9 mmol) in benzene (2000 ml) under nitrogen was addedN-bromo-succinimide (4.00 g, 22.5 mmol). The reaction mixture wasstirred just below reflux for 30 minutes, then at a gentle reflux for 30minutes. The resulting suspension was allowed to cool for 2 hours, thesolid filtered and dried at 70° C. under reduced pressure to give9-bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one (4.32 g, 83% purityby NMR).

[0096] E. Diethyl(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate

[0097] A solution of crude9-bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one (4.32 g), (S)-diethyl2-(5-amino-1-oxo-2-isoindolinyl) glutarate (4.0 g, 12 mmol), and NaHCO₃(2.0 g, 24 mmol) in DMF (30 ml) was stirred under nitrogen at 105° C.for 1.5 hours. After cooling, acetic acid (1 ml, 17 mmol) was added, thereaction mixture transferred to a larger round bottom flask withethanol, and then concentrated in vacuo onto silica gel (30 g). Theabsorbed material was purified by chromatography on silica gel elutingwith methanol:methylene chloride (1:24) and then precipitation of thesolid from methylene chloride (˜20 ml) with ethyl acetate (45 ml) andmethanol (5 ml). The white solid was filtered under nitrogen and driedunder high vacuum to give diethyl(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]-quinazolin-9-yl)-methyl)amino)-1-oxo-2-isoindolinyl)glutarate(3.27 g).

[0098] F(S)-2-(5-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid

[0099] A solution of diethyl(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxo-benzo-[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate(3.20 g, 5.75 mmol) in 0.2 N NaOH (140 ml) was stirred under nitrogenfor 3 hours at room temperature. The solution was then slowly adjustedto pH3 with 1 N HCl and the resulting suspension allowed to stirbriefly. The white solid was filtered under nitrogen, washed with waterand dried under high vacuum to give(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid (2.85). ¹H NMR (DMSO-d₆/D₂O, 300 MHz) δ: 1.86-2.34 (m, 4H, glu CH₂,CH₂), 2.44 (s, 3H, CH₃), 4.25 (very strongly coupled AB pair, 2H, gluNCH₂Ar), 4.58 (s, 2H, C⁹—CH₂), 4.67-4.75 (m, 1H, glu CH), 6.69-6.77 (m,2H, Ar), 7.37 (d, J=8 Hz, 1H Ar), 7.59 (d, J=9 Hz, 1H, Ar) 7.64 (dd,J=8.2 Hz 1H, Ar), 8.01 (d, J=8 Hz, 1H, Ar), 8.22 (d, J=9 Hz, 1H, Ar),9.85 (s, 1H, Ar). Anal Calculated for C₂₇H₂₄N₄O₆. 1.6H₂O: C, 61.27; H,5.18; N,10.58. Found: C 61.29; H, 5.18; N, 10.57.

EXAMPLE 2(s)-2-(5-(((3-Amino-1,2-dihydro-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid

[0100] A.N-(9-Bromomethyl-1,2-dihydro-1-oxobenzo[f]quinazolin-3-yl)pivalamide

[0101] N-(9-methyl-1,2-dihydro-1-oxobenzo[f]quinazolin-3-yl)pivalamide(15 g, 48 mmol) was dissolved in refluxing benzene (4000 ml). Thereaction was removed from heat and N-bromosuccinimide (11.28 g, 64 mmol,Kodak) added. The solution was heated under reflux for 2 hours. Benzenewas removed in vacuo, the residue slurried with a small volume ofethanol, filtered and dried under high vacuum to give the bromomethylderivative. The product was used without further purification.

[0102] BDiethyl(S)-2-(5-(((1,2-dihydro-1-oxo-3-pivalamidobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate(S)-diethyl 2-(5-amino-1oxo-2-isoindolinyl)glutarate (2.8) andN-(9-Bromomethyl-1,2-dihydro-1-oxobenzo[f]quinazolin-3-yl)pivalamide(2.0 g) were heated in dimethylformamide (15 ml) at 115° C. for 1.5hours. The reaction mixture was concentrated onto silica gel, elutingwith methylene chloride/methanol (97:3). The fractions containingproduct were evaporated and stored under high vacuum overnight. Thepartially crystallized residual oil was suspended in ethyl acetate andfiltered. The solid was recrystallized from methanol, filtered and driedunder high vacuum to yield the diester (0.21 g) as a white solid.

[0103] C.(S)-2-(5-(((3-Amino-1,2-dihydro-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid

[0104] A solution of diethyl(S)-2-(5-(((1,2-dihydro-1-oxo-3-pivalamidobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutarate in 0.33M aqueous sodium hydroxide/tetrahydrofuran (12 ml, 1:1) was heated toreflux. Additional water (6 ml) was added to keep the reaction mixturehomogeneous, and the solution was heated under reflux for 2.5 hours. Thecooled solution was adjusted to pH 3 with 1M hydrochloric acid, theprecipitate filtered off, washed with water and dried under high vacuum.The crude product, a white solid (0.15 g), contained approximately 20%of the 3-pivalamide of the desired diacid by NMR. The solid wasredissolved in 0.5 M sodium hydroxide (5 ml) and stirred at roomtemperature for 2 days. The pH of the solution was adjusted to 2, theprecipitated-solid filtered off, washed with water, and dried at roomtemperature under high vacuum to yield the diacid as a white solid (0.13g). ¹H NMR (DMSO-d₆, 300 MHz); δ1.88-2.08 (m, 1H), 2.10-2.30 (m,3H),2.26 (s, 2H), 4.52 (d, J=5.6 Hz, 2H), 4.65-4.75 (m, 1H), 6.57 (br s,2H), 6.67-6.75 (overlapping s and dd, 2H), 7.13 br t, J=5.6 Hz, 1H),7.27 (d, J=8.9 Hz, 1H) 7.35 (d, J=8.3 Hz, 1H), 7.46 (dd, J=8.3, 1.1 Hz,1H), 7.85 (d,j=8.2 Hz, 1H), 8.00 (d, J=8.9 Hz, 1H), 9.68 (s, 1H), 11.14(brs, 1H), 11.9-12.9 (v br s 2H. Anal. Calculated for C₂₈H₂₃N₅O₆.2.5H₂O: C. 57.61 H, 4.93; N, 12.80.

EXAMPLE 3N-4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L-glutamicacid

[0105] A.Diethyl-N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)sulfonamido)benzoyl)-L-glutamate

[0106] N-(4-aminobenzoyl)-L-glutamic acid diethyl ester (6.06 g, 0.0188mole) (Aldrich) and1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]-quinazolin-9-sulfonyl chloride(5.84 g, 0.0188 mole) were dissolved in pyridine (55 ml) and thereaction mixture stirred at room temperature for 3.5 hours. The pyridinewas removed in vacuo, the residue washed with water, and the pink solidcollected by filtration. The crude product was dried under high vacuum,then subjected to chromatography on a Waters Prep 500 instrument (silicaCartridge, elution with methanol: methylene chloride (1:4). The productwas recrystallized from ethanol and dried under high vacuum to yield thediethyl ester (5.68 g, 51%).

[0107] B.N-(4-((1,2,5,6-Tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)-sulfonamido)benzoyl)-L-glutamicacid

[0108] Diethyl-N-(4-((1,2,5,6-tetrahydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)sulfonamido) benzoyl)-L-glutamate (4.53 g, 7.6 mmole)was dissolved in N-NaOH (64 ml) and the solution stirred at roomtemperature for 4 hours. The pH of the solution was adjusted to 3.00with 1N HCl, the solid collected by filtration, washed with water anddried under high vacuum to yield the product as an off-white solid (3.94g 96%). ¹HNMR (DMSO-d₆,300 MHz) δ: 1.84-1.96 (m,1H,glu CH); 2.00-2.10(m, 1H, glu CH); 2.32(s, 3H, CH₃, superimposed over t, 2H, glu CH₂);2.71 (m, 2H, Ar CH₂); 2.89 (m, 2H, Ar CH₂); 4.28-4.36 (m, 1H, glu CH),7.20 (d, J=9 Hz, 2H, Ar); 7.39 (d, J=8 Hz, 1H, Ar); 7.62 (dd, J=8.2 Hz,1H, Ar); 7.74 (d, J=9Hz, 2H, Ar); 8.44 (d, J=8 Hz, 1H, gluNH); 9.21 (d,J=2Hz, 1H, Ar); 10.73 (s, 1H, SO₂NH); 12.36 (br s, 2H, CO₂H); 12.72 (brs, 1H, NH). Anal Calculated for C₂₅H₂₄N₄O₈S. 3/2H₂O: C,52.91; H,4.79;N,9.87; S,5.65. Found: C,52.98; H,4.78; N,9.87; S,5.58.

EXAMPLE 4N-(4-(((1,2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)-amino)-2-fluorobenzoyl)-L-glutamicacid

[0109] A. DiethylN-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-fluorobenzoyl)-L-glutamate

[0110] To a hot solution of 3,9-dimethylbenzo[f]quinazolin-1(2H)-one(2.0 g, 8.9 mmol) in benzene (1000 ml) under nitrogen was addedN-bromosuccinimide (NBS) (2.0 g, 11 mmol). The solution was stirred atreflux for 1 hour and then concentrated in vacuo to give crude9-bromomethyl-3-methylbenzo[f]quinazolin-1(2H)-one. The solid wassuspended with diethyl N-(4-amino-2-fluorobenzoyl)-L-glutamate (T. R.Jones et al., UK Patent GB 2175 903A, 1986)(6.0 g, 18 mmol) in DMF (20ml) and stirred under nitrogen at 100° C. for 30 minutes. The reactionmixture was allowed to cool. N-methylmorpholine (1.0 ml, 9.1 mmol)(Aldrich) was added, and the solution concentrated under high vacuum.The residue was purified with silica gel chromatography eluting withmethylene chloride:THF (5:1). Fractions containing product wereconcentrated in vacuo to a thick paste, the solid suspended in a smallvolume of diethyl ether, filtered under nitrogen, and dried under highvacuum to give diethylN-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]-quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamateas a white solid (2.3 g).

[0111] B.N-(4-(((2-Dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)-amino)-2-fluorobenzoyl)-L-glutamicacid

[0112] A solution of diethylN-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamate(2.3 g, 4.1 mmol) in ethanol (25 ml) and 0.2 N NaOH (100 ml) was stirredunder nitrogen at room temperature for 3 hours. The solution wasadjusted to pH 7 with 1N HCl and reduced in volume under vacuum toremove the ethanol. The product was precipitated by acidifying thesolution with 1N HCl to pH 3 with stirring under nitrogen. Thesuspension was stirred 15 minutes, filtered under nitrogen, washed withwater, pressed with a sheet of latex to remove excess water, and driedunder high vacuum to giveN-(4-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quina-zolin-9-yl)methyl)amino)-2-fluorobenzoyl)-L-glutamicacid as a white solid (2.1 g. ¹H NMR (DMSO-d₆,300 MHz) d: 1.82-2.12 (m,2H, glu CH₂), 2.29 (t, J=7 Hz, 2H, glu CH₂), 2.43 (s, 3H, C³—CH₃),4.32-4.42 (m, 1H, glu CH), 4.57 (d, J=6 Hz, 2H, C⁹—CH₂), 6.39 (dd,J=15.2 Hz, 1H, Ar), 6.53 (dd, J=9.2 Hz 1H, Ar), 7.30 (t, J=6 Hz, 1H,ArNH), 7.47 (dd, J=9.9 Hz, 1H, Ar), 7.59(d, J=9 Hz, 1H. Ar), 7.61(dd,J=8.2 Hz, 1H, Ar), 7.73 (t, J=7 Hz, 1H, glu NH), 8.01 (d, J=8 Hz, 1H,Ar), 8.22 (d, J=9 Hz, 1H, Ar), 9.85 (s, 1H, Ar), 12.42 (br s, 2H,CO₂H's), 12.53 (s, 1H, N²H) Anal. Calculated for C₂₆H₂₃FN₄O₆.3/2 H₂O.1/3NaCl: C, 56.49: H, 4.74: N, 10.14; Cl, 2.12: Na, 1.37. Found: C, 56.48:H, 4.64; N, 10.20: Cl, 2.01; Na, 1.30.

EXAMPLE 5

[0113] The thymidylate synthase inhibitor Compound A (8 mg/kg) waslethal when administered to beagle dogs as an iv bolus for fiveconsecutive days. In contrast Compound A at 20 mg/kg was not lethal whengiven as an iv bolus 30 minutes after oral administration of folic acidat 50 mg/kg. Similarly, five consecutive days of 10 mg/kg of Compound Aiv was lethal when coven alone. But when the same dose was given 30minutes after an oral dose of 10, 50, 100 or 200 mg/kg of leucovorin(Wellcovorin) no deaths occurred.

EXAMPLE 6

[0114] Compound A administered as an ip bolus for five days at 3.2 or 10mg/kg to nude mice bearing the human tumor GC3TK in the subrenal capsuleproduced complete inhibition of tumor growth. When 50 mg/kg of folicacid or 200 mg/kg leucovorin was administered orally 30 minutes prior tothe ip bolus dose of Compound A complete inhibition of tumor growth wasstill observed. In another experiment, in the absence of protectant 10mg/kg Compound A produced complete growth inhibition and 3.2 mg/kgproduced 89% inhibition. When 500 mg/kg folic acid was administeredorally 30 min prior to the Compound A, tumor growth inhibition was notsignificantly different from that observed in the absence of the folicacid. TABLE Influence of Folic Acid or leucovorin upon toxicity andantitumor efficacy of Compound A [Folic Acid] mg/kg) 0 10 50 100 200 50010 mg/kg Compound A lethal +/− m m m m Dog tox. bone marrow toxicityShort term: — — Long term: — 10 mg/kg Compound A >100 >100 Mouseantitumor (% I) 3.2 mg/kg Compound A  89  78 Mouse antitumor (% I) 10mg/kg Compound A >100 >100 Mouse antitumour (% I) 3.2 mg/kg Compound A 100  100 Mouse antiumor (% I) [leucovorin] (mg/kg) 0 5 10 50 100 200 10mg/kg Compound A Lethal +/− m m m m Dog tox bone marrow toxicity — —Short term: 10 mg/kg Compound A >100 >100 Mouse antitumor (% I) 3.2mg/kg Compound A  100  100 Mouse antitumor (% I)

We claim:
 1. A method of reducing intestinal toxicity associated withthe administration of a therapeutically effective amount of anon-competitive thymidylate synthase inhibitor to a mammal, comprising:administering to said mammal a folate derivative selected from folicacid, 5-formyltetrahydrofolate, or 5-methyltetrahydrofolate in an amounteffective to reduce said toxicity without blocking therapeutic effect ofthe non-competitive thymidylate synthase inhibitor, wherein thenon-competitive thymidylate synthase inhibitor is(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid.
 2. A method as claimed in claim 1, wherein the folate derivativeis folic acid.
 3. A method as claimed in claim 1, wherein the folatederivative is administered either, before, concurrentit, with, or afterthe administration of the thymidylate synthase inhibitor.
 4. A method ofreducing intestinal toxicity associated with the administration of atherapeutically effective amount of a non-competitive thymidylatesynthase inhibitor to a mammal, comprising: administering to said mammalfolic acid in an amount effective to reduce said toxicity withoutblocking therapeutic effect of the non-competitive thymidylate synthaseinhibitor, wherein the non-competitive thymidylate synthase inhibitor is(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid.
 5. A method of treating a susceptible tumor in a mammal,comprising: administering to said mammal a therapeutically effectiveamount of(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid and folic acid in an amount effective to reduce said toxicitywithout blocking therapeutic effect of the(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid.
 6. A cancer treatment combination for treating a susceptible tumorin a mammal, comprising: a therapeutically effective amount of(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid and folic acid in an amount effective to reduce said toxicitywithout blocking therapeutic effect of the(S)-2-(5-(((1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl)amino)-1-oxo-2-isoindolinyl)glutaricacid.